12Background 13 Bicyclic boronates are a new and potentially important class of β-lactamase inhibitor, 14 with the ability to inhibit β-lactamases from all molecular classes, including mobile 15 metallo-β-lactamases.
16Objective 17 Our objective was to identify mutants resistant to the actions of the bicyclic boronate 18 inhibitor 2, when being used in combination with aztreonam.
19
Methods
20Overnight cultures were plated on to agar containing increasing concentrations of 21 aztreonam with a fixed 10 mg/L concentration of the inhibitor. Resistant derivatives 22 and parent strains were analysed by whole genome sequencing and LC-MS/MS 23 proteomics to identify mechanism of resistance.
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Results
25When using a mixed overnight culture containing one Escherichia coli (TEM-1, CTX-26 M-15, CMY-4 producer) and one Klebsiella pneumoniae (SHV-12, CTX-M-15, NDM-27 1 producer) mobilisation of an IncX3 plasmid carrying blaSHV-12 from the K. 28 pneumoniae into the E. coli generated an aztreonam/boronate resistant derivative. 29 Conclusions 30 High-level production of three bicyclic boronate susceptible enzymes (CMY-4, CTX-31 M-15, SHV-12) capable of hydrolysing aztreonam plus TEM-1, which binds the 32 inhibitor, overcomes the fixed inhibitor dose used. This was only identified when 33 3 using a mixed culture for selection. It would seem prudent that to allow for 34 coalescence of the myriad β-lactamase genes commonly found in bacterial 35 populations colonising humans, this mixed culture approach should be the norm 36 when testing the potential for generating β-lactamase inhibitor resistance in pre-37